Project Summary: Dental caries is one of the most prevalent and familiar forms of infectious disease afflicting humans. Our understanding of the disease and its treatments has improved dramatically over the past half-decade through the use of rodent caries models and ever-improving experimental approaches. However, the physical mechanisms and structural changes within enamel during the initiation of caries are still poorly understood. Specifically, the dynamic evolution of the outermost, intact surface zone remains a relative mystery due to challenges of performing in situ investigations in humans and the hierarchical structural and compositional complexity of enamel. It is the goal of the proposed research to both address the immediate knowledge gap regarding in vivo surface zone evolution and develop a microfluidics platform to enable unprecedented characterization at high spatial and temporal resolution during in vitro demineralization. In aims 1 and 2, a controlled sequence of non-destructive characterization techniques will be applied at increasingly smaller scales such that results can be correlated for each individual surface zone examined. Furthermore, I recently demonstrated the ability to generate surface zone lesions in a rodent caries model, providing much finer experimental control over lesion formation and synchronization when compared to humans. By applying this model to generate a large number of surface zones and then characterizing them across length scales, I will provide a more complete picture of the structural and compositional evolution of the surface zone and how this process is influenced by fluorine in the oral cavity. Finally, to address the challenges to obtaining high time resolution information regarding SZ evolution, I intend to develop a novel microfluidics platform that enables transmission-based characterization of in situ demineralization of thin enamel sections in aim 3. This will allow direct observation of in vitro lesion development at high temporal and spatial resolution for the first time. All together, this research will dramatically improve our understanding of and future ability to probe the mechanisms of caries initiation. The knowledge gained will improve the treatment of early-stage caries and inform the development of novel preventative treatments.